Gene expression, which is for biological processes such as aging, development, differentiation, metabolite production, progression of the cell cycle, and infectious or genetic or other disease states, can be characterized by determining the level (i.e., the concentration of mRNA in a sample, etc.) or pattern (i.e., the kinetics of expression, rate of decomposition, stability profile, etc.) of the expression of a protein encoded by a gene under study. A variety of methods are currently available for accomplishing this task. One such method is a microarray-based approach (Schena et al., Science 270: 467-470 (1995), which is incorporated herein by reference in its entirety). This ‘chip’-based approach involves: (1) generating two populations of fluorescently labeled cDNA probes from two MRNA samples which are isolated from two physiological sources, where each population of cDNA probes is labeled with one of two distinct labeled oligonucleotides capable of generating distinguishable fluorescent signals at two wavelengths; (2) simultaneously hybridizing the two populations of fluorescently labeled cDNA probes to gene targets on a microarray; (3) separately detecting intensities of hybridization signals for each of elements on the microarray at two distinguishable wavelengths and determining the hybridization patterns of two samples; and (4) calculating ratios of gene expression levels from two co-hybridized samples. This ‘chip’-based technology can allow quantitatively monitoring of differential expression of hundreds and thousands of genes simultaneously (Schena et al., Proc. Natl. Acad. Sci. USA 93: 10614-10619 (1996); Heller et al., Proc. Natl. Acad. Sci. USA 94: 2150-2155 (1997); DeRisi et al., Science 278: 680-686; and Ruan et al., Plant J. 15: 821-833 (1998), all of which are incorporated herein by reference in their entirety) and can provide a powerful tool for gene discovery, functional analysis and elucidation of genetic regulatory networks.
Despite the great promise of this ‘chip’-based technology, there are still problems with respect to enhancing its reliability and optimizing its performance. For instance, its detection sensitivity still needs to be greatly improved. The detection sensitivity of the microarray technology is mainly limited by the fluorescence emission quantum yield of labeled oligonucleotides for labeling cDNA probes. With currently available labeled oligonucleotides comprising a single fluorophore, hybridization signals for low expressed genes could be so weak that the intrinsic noise of instruments could render ratio measurements unreliable. Without further enhancement in signal-to-noise ratio, current microarray methods probably will be limited to the characterization of differential expression for highly-expressed genes.
One class of labeled oligonucleotides which typically has been developed is energy transfer (ET) fluorescent dyes. Such energy transfer fluorescent dyes include a donor fluorophore and an acceptor fluorophore. In these dyes, when the donor and acceptor fluorophores are positioned in proximity with each other and with the proper orientation relative to each other, the energy emission from the donor fluorophore is absorbed by the acceptor fluorophore and causes the acceptor fluorophore to emit light at a longer wavelength. In these ET labeled oligonucleotides, such energy transfer was exploited to make a set of labels having high absorbance at a common wavelength but well-separated fluorescence emission maximums. However, the fluorescence per ET label molecule was not increased. Such ET labeled oligonucleotides are not suitable for enhancing the detection sensitivity of the microarray technology, since two distinct labeled oligonucleotides can be excited at two distinguishable wavelengths.
Thus, there remains a continuing need for new labeled oligonucleotides which have much higher fluorescence per molecule and could enhance greatly the signal to noise ratio so that differential expression of low expressed genes could be reliably determined.
Furthermore, current methods of fluorescently labeled cDNA preparations are not entirely satisfactory. In current preparation methods, poly A RNA (mRNA) needs to be isolated first from total RNA and then fluorescent cDNA probes are prepared from the isolated mRNA. One problem with current methods is that large amounts of starting materials are required because of potential sample loss during mRNA isolation. Another problem is that the lost amount of a specific MRNA can vary from preparation to preparation from the above two laborious preparation steps. Such preparation-to-preparation variation can artificially introduce apparent differential expression of genes being tested and affect the reliability of the microarray method. Another problem is that current methods can generate fluorescently labeled cDNA probes of short length, for instance, less than about 300 nucleotides. With such short probes, hybridization of the cDNA probes to the targets on microarrays has to be carried out under less stringent conditions so that the reliability of experimental results is compromised. If the length of cDNA probes ranged from 500 to 1200 nucleotides, the hybridization of the fluorescent cDNA probes to the targets on microarrays could be performed under optimal conditions.
Thus, there remains a continuing need for the development of cDNA probe preparation methods wherein the amount of starting materials and artificial errors in ratios of gene expression levels can be minimized, the length of cDNA probes is longer, and the cDNA probes have much higher fluorescence per molecule.
It is an object of the present invention to provide a labeled oligonucleotide which has high fluorescence per molecule and can be used to detect biological molecules with high sensitivity.
It is another object of the invention to provide an oligonucleotide primer which includes the labeled oligonucleotide of the invention.
It is another object of the invention to provide a method for generating, from smaller amount of RNA samples, fluorescently labeled cDNA probes which have a much higher fluorescence per molecule and the length of which is from 500 to 1000 nucleotides.